Abstract
The eukaryotic replication factor C (RFC) clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the delta clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2.3.4.5 and Rfc2.5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli delta wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad.RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad.RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.
Highlights
The proliferating cell nuclear antigen (PCNA)3 DNA sliding clamp is a ring-shaped homotrimer that is opened and closed around DNA by the replication factor C (RFC) clamp loader [1,2,3,4]
All Five RFC Subunits Bind PCNA—Proteins that bind sliding clamps often do so via hydrophobic interaction in a binding pocket positioned between two globular domains of the clamp
This was initially demonstrated for p21CIP1/WAF1 cell cycle inhibitor binding to human PCNA via the conserved PCNA-interacting peptide sequence within p21CIP1/WAF1 [3]
Summary
Nomenclature and arrangement of clamp loader subunits from different organisms The five subunits of the clamp loader are arranged in a spiral, with each position designated by a letter. Clamp loading buffer contained 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 40 g/ml bovine serum albumin, and 5% glycerol. Gel filtration buffer was composed of 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 100 g/ml bovine serum albumin, 8 mM MgCl2, 4% glycerol, and 150 mM NaCl. Rfc4(B) is positioned over an interface and contacts PCNA in a minimal fashion; Rfc3(C) binds the second protomer of PCNA, but buries less surface area than the Rfc1-PCNA contact; and Rfc2(D) and Rfc5(E) are suspended above PCNA and do not bind the ring at all. The Rfc21⁄731⁄741⁄75 subcomplex of the four small RFC subunits can open PCNA clamps and clear them from DNA. These internal workings mark a significant departure in the underlying mechanism of clamp opening by eukaryotic and prokaryotic clamp openers
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